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Bacterially formed microcin s, a new antimicrobial peptide, effective against pathogenic microorganisms, e.g. enterohemorrhagic escherichia coli (EHEC)

a technology of microcin and sulfonamide, which is applied in the field of isolated polypeptides and nucleic acid molecules encoding microcin s polypeptides, can solve the problems of increasing the difficulty of esbl therapy, the emergence of pathogens resistant to classical antibiotics, and the inability to induce allergies, etc., to achieve the effect of not inducing allergies

Inactive Publication Date: 2017-01-12
SYMBIOGRUPPE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0049]That is, the present inventors have identified the polypeptide microcin S which is a new, not previously described microcin, whose nucleotide and amino acid sequence is unique. The genes which are responsible for the effects of microcin S have been identified and their effects have been functionally characterized. The inventors have surprisingly found that, in contrast to most of the classical beta-lactam-antibiotics, microcin S shows an inhibitory effect of EHEC including the ESBL-producing O104:H4 outbreak strain as well as an O128:H2 and an O157:H7 EHEC isolate (FIG. 5). Thus, it has been further found that microcin S is effective in the treatment of enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) infection. Therefore, microcin S might be used as an alternative to conventional antibiotics and thus to the control of pathogenic microorganisms. Furthermore, a use for food preservation is possible, and the present invention was thereby completed. It has been further found that the microcin S (MccS) gene cluster (SEQ ID NO: 1) of E. coli G3 / 10, encoded by plasmid pSYM1 (FIG. 1), consists of the four clustered genes mcsS (SEQ ID NO: 2), mcsI (SEQ ID NO: 3), mcsA (SEQ ID NO: 4) and mcsB (SEQ ID NO: 5). Since E. coli G3 / 10 is a well-proven probiotic used for example for the treatment of gastrointestinal disorders, microcin S does not necessarily have to be purified from the strain. MccS should advantageously be effective in vivo.
[0050]Advantages are that the microcin S is nontoxic, may not induce allergies, and is difficult for pathogenic microbials to develop resistance against.
[0051]Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Problems solved by technology

However, the emergence of pathogens resistant to these classical antibiotics constitutes a massive problem.
Consequently, the remaining treatment options are even more restricted.
Therapy of enhanced-spectrum beta-lactamase (ESBL) strains is increasingly challenging.

Method used

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  • Bacterially formed microcin s, a new antimicrobial peptide, effective against pathogenic microorganisms, e.g. enterohemorrhagic escherichia coli (EHEC)
  • Bacterially formed microcin s, a new antimicrobial peptide, effective against pathogenic microorganisms, e.g. enterohemorrhagic escherichia coli (EHEC)
  • Bacterially formed microcin s, a new antimicrobial peptide, effective against pathogenic microorganisms, e.g. enterohemorrhagic escherichia coli (EHEC)

Examples

Experimental program
Comparison scheme
Effect test

example 1

Bacterial Strains and Growth Conditions

[0217]Bacterial strains used herein are listed in Table 3.

TABLE 3Bacterial Strains UsedRelevantStrainGenotypeOriginE. coli Nissle 1917wild-typeMutaflor ®E. coli G1 / 2awild-typeSymbioflor 2 ®,SymbioPharmE. coli G3 / 103wild-typeSymbioflor 2 ®,SymbioPharmE. coli G4 / 9awild-typeSymbioflor 2 ®,SymbioPharmE. coli G5awild-typeSymbioflor 2 ®,SymbioPharmE. coli G6 / 7awild-typeSymbioflor 2 ®,SymbioPharmE. coli G8awild-typeSymbioflor 2 ®,SymbioPharmE. coli MDS42K-12 multiplePosfai et al. (2006)deletion strainEmergent Properties ofReduced-GenomeEscherichia coli.Science 312:1044-1046.EPEC E2348 / 69 O127:H6Ampr (pUC19),Donnenberg & KaperKanar (pUC4k)(1992) Enteropathogenicor CmrEscherichia coli. Infect.(pACYC184)Immun. 60: 3953-3961.EHEC 86-24 O157:H7stx2, eaeA,Griffin et al. (1988)EHEC-hlyA,Illnesses associated withastA, SmrEscherichia coli 0157:H7infections. A broadclinical spectrum.Ann. Intern. Med.109: 705-712.EHEC O104:H4wild-type, stx2,human isolate, Dresde...

example 2

Identification of a Microcin-Encoding Gene Cluster in E. coli G3 / 10

[0218]The genomes of six E. coli: E. coli G1 / 2, G3 / 10, G4 / 9, G5, G6 / 7 and G8. were sequenced. Annotation revealed no known microcin within the genome of E. coli G3 / 10. E. coli G3 / 10 contains a large conjugative plasmid pSYM1 (FIG. 1) with a size of 50.6 kb. The plasmid is 99% identical to plasmid pMAS2027 of an uropathogenic E. coli isolate. Moreover, it contains a 10 kb insertion fragment, but BLAST analysis revealed only uncharacterized and unnamed genes. To identify the origin of bactericidal action it was tried to cure the strain E. coli G3 / 10 from its megaplasmid pSYM1. Despite performing many of common curing procedures, for example mitomycin C or heat treatment, the strain could not be cured. Therefore, plasmid pSYM1 was transferred to E. coli G4 / 9 by conjugation. To allow a screening of conjugants, at first an ampicillin resistance cassette was integrated into pSYM1 resulting in pSYM1-ST76An. The resulting E....

example 3

Functional Characterization of Microcin S and Elucidating its Self Immunity

[0219]Bacterial adhesion is a crucial first step of many infectious diseases. Therefore, a test system quantifying the inhibition of adherence to human intestinal epithelial cells is suitable to demonstrate a beneficial effect to the host. In a first experiment, confluent monolayers of CACO-2 or LOVO cells were pre-incubated with bacterial test strains EcN, E. coli G3 / 10, E. coli G4 / 9 or E. coli G4 / 9 pAZ6 at an MOI of 100:1 E. coli to host cells. After two hours of incubation, cells were washed and infected with EPEC E2348 / 69 using an MOI of 100:1 EPEC to host cells. E. coli G3 / 10 and E. coli G4 / 9 pAZ6 were capable of inhibiting EPEC adherence similar to EcN. EcN adherence inhibition was shown to be dependent on the strains microcins activity. Adherence efficiency in % are expressed as adherence of EPEC relative to adherence without any pre-incubation (negative control), which is set 100%. The data are the me...

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Abstract

The present invention relates to a new isolated polypeptide nominated microcin S, isolated nucleic acid molecules encoding the microcin S polypeptide and primers and probes hybridizing to the nucleic acid molecules. The invention also relates to plasmids and cells comprising the nucleic acid molecules, an antibody binding to the polypeptide, compositions as well as methods for producing and using the polypeptides. The present invention further relates to medical uses for treating or preventing microbial infections, functional gastrointestinal disorders or treating a tumor. The invention further relates to a method for preserving food and a method for coating dressing material.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a continuation of U.S. patent application Ser. No. 14 / 238,370, filed Feb. 11, 2014 (now pending), which itself is a U.S. National Stage application of PCT International Patent Application Serial No. PCT / EP2012 / 065804, filed Aug. 13, 2012, which itself claims the benefit European Patent Application Serial No. EP 11 177 451.9, filed Aug. 12, 2011. The disclosure of each of these applications is incorporated herein by reference in its entirety.TECHNICAL FIELD[0002]The present invention relates to an isolated polypeptide, isolated nucleic acid molecules encoding a microcin S polypeptide and primers and probes hybridizing to the nucleic acid molecules. The invention also relates to plasmids and cells comprising the nucleic acid molecules, an antibody binding to the polypeptide, compositions as well as methods for producing and using the polypeptides. The present invention further relates to medical uses for treating or preve...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/245A23L3/3463A23B4/22A23C3/08A01N63/00A61K9/70A01N63/50
CPCC07K14/245A01N63/00A61K9/70A23V2002/00A23C3/08A23L3/34635A61K38/00A23B4/22A01N47/44A01N57/16C07K16/1232A61P1/00A61P1/04A61P17/00A61P31/02A61P31/04A61P35/00Y02A50/30A01N63/50C12N15/00C12Q1/00
Inventor GUNZER, FLORIANBIELACK, ANKEZIMMERMANN, KURT
Owner SYMBIOGRUPPE
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